Endothelial dysfunction defined as a loss of biologically active nitric oxide (NO) produced in the endothelium, is one of the most important events in initiation and progression of vascular disease. Tetrahydrobiopterin (BH4) is an essential cofactor needed for enzymatic activity of nitric oxide synthase(s) and biosynthesis of NO. In vivo mechanisms responsible for the control of BH4 metabolism are poorly understood. During preliminary studies for this application we identified erythropoietin (EPO) as a novel and potent stimulator of BH4 biosynthesis in the cardiovascular system. EPO is circulating hormone responsible for control of erythropoiesis, however, more recent findings suggest that EPO has important non-erythropoietic effects including vascular protection. The general hypothesis of this proposal is that in vivo, BH4 critically contributes to the vascular protective effect of EPO. To test this hypothesis we propose studies with following specific aims: 1) determine the role of BH4 in mediation of protective effectsof EPO in vascular endothelium, 2) determine if EPO may prevent development of atherosclerosis, and 3) analyze the mechanisms of EPO- induced endothelial repair after vascular injury. In vivo genetic and pharmacological approaches will be used to manipulate levels of circulating EPO and BH4. Established murine models of endothelial dysfunction and vascular injury will be employed to determine role of BH4 in mediation of vascular protective effects of EPO. To dissect direct effects of EPO from its rheological effects due to increase in shear stress imposed on endothelium by circulating erythrocytes, effects of novel non-erythropoietic derivatives of EPO will be studied. It is anticipated that the results of the proposed experiments will provide novel and important information concerning the role of EPO in control of BH4 metabolism in the cardiovascular system. This information may help to develop new strategies in prevention and treatment of endothelial dysfunction. .